Stabilized P53 peptides and uses thereof

ABSTRACT

Cross-linked peptides related to human p53 and bind to HMD2 or a family member of HDM2 useful for promoting apoptosis, e.g., in the treatment of and identifying therapeutic agents that binding to HMD2 or a family member of HDM2.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.14/483,905, filed Sep. 11, 2014, which is a continuation of U.S.application Ser. No. 12/525,123, filed Mar. 18, 2010, now U.S. Pat. No.8,889,632, which was the National Stage of International Application No.PCT/US2008/052580, filed Jan. 31, 2008, which claims the benefit of U.S.Provisional Application Ser. No. 60/887,526, filed Jan. 31, 2007, eachof which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 18, 2014, isnamed 35224-751.831_SL.txt and is 31,675 bytes in size.

The human p53 transcription factor induces cell cycle arrest andapoptosis in response to DNA damage¹ and cellular stress,² therebyplaying a critical role in protecting cells from malignanttransformation. The E3 ubiquitin ligase HDM2 controls p53 levels througha direct binding interaction that neutralizes p53 transactivationactivity, exports nuclear p53, and targets it for degradation via theubiquitylation-proteasomal pathway.^(3, 4) Loss of p53 activity, eitherby deletion, mutation, or HDM2 overexpression, is the most common defectin human cancer.⁵ Tumors with preserved expression of wild type p53 arerendered vulnerable by pharmacologic approaches that stabilize nativep53. In this context, HDM2 targeting has emerged as a validated approachto restore p53 activity and resensitize cancer cells to apoptosis invitro and in vivo.⁶ HDMX (HDM4) has also been identified as a regulatorof p53. Moreover, studies have shown a similarity between the p53binding interface of HDM2 and that of HDMX.^(6a)

The p53-HDM2 protein interaction is mediated by the 15-residue α-helicaltransactivation domain of p53, which inserts into a hydrophobic cleft onthe surface of HDM2.⁷ Three residues within this domain (F19, W23, andL26) are essential for HDM2-binding.^(8, 9)

SUMMARY

Described below are stably cross-linked peptides related to a portion ofhuman p53 (“stapled p53 peptides”). These cross-linked peptides containat least two modified amino acids that together form an internalcross-link (also referred to as a tether) that can help to stabilize thealpha-helical secondary structure of a portion of p53 that is thought tobe important for binding of p53 to HDM2. Accordingly, a cross-linkedpolypeptide described herein can have improved biological activityrelative to a corresponding polypeptide that is not cross-linked. Thestapled p53 peptides are thought to interfere with binding of p53 toHDM2 thereby inhibiting the destruction of p53. The stapled p53 peptidedescribed herein can be used therapeutically, e.g., to treat a varietyof cancers in a subject. For example, cancers and other disorderscharacterized by an undesirably low level or a low activity of p53and/or cancers and other disorders characterized by an undesirably highlevel of activity of HDM2. The modified peptides may also be useful fortreatment of any disorder associated with disrupted regulation of thep53 transcriptional pathway, leading to conditions of excess cellsurvival and proliferation (e.g., cancer and autoimmunity), in additionto conditions of inappropriate cell cycle arrest and apoptosis (e.g.,neurodegeneration and immune deficiency).

In one aspect, the invention features a modified polypeptide of Formula(I),

or a pharmaceutically acceptable salt thereof,

wherein;

each R₁ and R₂ are independently H or a C₁ to C₁₀ alkyl, alkenyl,alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, orheterocyclylalkyl;

R₃ is alkylene, alkenylene or alkynylene, or [R₄′—K—R₄]_(n); each ofwhich is substituted with 0-6 R₅;

R₄ and R₄′ are independently alkylene, alkenylene or alkynylene (e.g.,each are independently a C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10alkylene, alkenylene or alkynylene);

R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;

K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

aziridine, episulfide, diol, amino alcohol;

R₆ is H, alkyl, or a therapeutic agent;

n is 2, 3, 4 or 6;

x is an integer from 2-10;

w and y are independently an integer from 0-100;

z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and

each Xaa is independently an amino acid (e.g., one of the 20 naturallyoccurring amino acids or any naturally occurring non-naturally occurringamino acid);

wherein the polypeptide, comprises at least 8 contiguous amino acids ofSEQ ID NO:1 (human p53) or a variant thereof, SEQ ID NO:2 or a variantthereof, or another polypeptide sequence described herein except that:(a) within the 8 contiguous amino acids of SEQ ID NO:1 the side chainsof at least one pair of amino acids separated by 3, 4 or 6 amino acidsis replaced by the linking group, R₃, which connects the alpha carbonsof the pair of amino acids as depicted in Formula I; and (b) the alphacarbon of the first of the pair of amino acids is substituted with R₁ asdepicted in formula I and the alpha carbon of the second of the pair ofamino acids is substituted with R₂ as depicted in Formula I.

In another aspect, the invention features a modified polypeptide ofFormula (II),

or a pharmaceutically acceptable, salt thereof,

wherein;

each R₁ and R₂ are independently H or a C₁ to C₁₀ alkyl, alkenyl,alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, orheterocyclylalkyl;

R₃ is alkylene, alkenylene or alkynylene, or [R₄′—K—R₄]_(n); each ofwhich is substituted with 0-6 R₅;

R₄ and R₄′ are independently alkylene, alkenylene or alkynylene (e.g.,each are independently a C1, C2, C3, C4, C5, C6, C7, C8, C9 or C10alkylene, alkenylene or alkynylene);

R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, afluorescent moiety, or a radioisotope;

K is O, S, SO, SO₂, CO, CO₂, CONR₆, or aziridine, episulfide, diol,amino alcohol;

R₆ is H, alkyl, or a therapeutic agent;

n is 2, 3, 4 or 6;

x is an integer from 2-10;

w and y are independently an integer from 0-100;

z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and

each Xaa is independently an amino acid (e.g., one of the 20 naturallyoccurring amino acids or any naturally occurring non-naturally occurringamino acid);

R₇ is PEG, a tat protein, an affinity label, a targeting moiety, a fattyacid-derived acyl group, a biotin moiety, a fluorescent probe (e.g.fluorescein or rhodamine) linked via, e.g., a thiocarbamate or carbamatelinkage;

R₈ is H, OH, NH₂, NHR_(8a), NR_(8a)R_(8b);

wherein the polypeptide comprises at least 8 contiguous amino acids ofSEQ ID NO:1 (human p53) or a variant thereof, SEQ ID NO:2 or a variantthereof, or another polypeptide sequence described herein except that:(a) within the 8 contiguous amino acids of SEQ ID NO:1 the side chainsof at least one pair of amino acids separated by 3, 4 or 6 amino acidsis replaced by the linking group, R₃, which connects the alpha carbonsof the pair of amino acids as depicted in formula I; and (b) the alphacarbon of the first of the pair of amino acids is substituted with R₁ asdepicted in Formula II and the alpha carbon of the second of the pair ofamino acids is substituted with R₂ as depicted in Formula II.

In the case of Formula I or Formula II, the following embodiments areamong those disclosed.

In cases where x=2 (i.e., i+3 linkage), R₃ can be a C7 alkylene,alkenylene. Where it is a alkenylene there can one or more double bonds.In cases where x=6 (i.e., i+4 linkage), R₃ can be a C11, C12 or C13alkylene or alkenylene. Where it is a alkenylene there can one or moredouble bonds. In cases where x=3 (i.e., i+4 linkage), R₃ can be a C8alkylene, alkenylene. Where it is a alkenylene there can one or moredouble bonds.

SEQ ID NO:1 is the sequence of human p53. The stapled peptides caninclude the sequence Leu Ser Gln Glu Thr Phe Ser Asp Leu Trp Lys Leu LeuPro Glu Asn (SEQ ID NO:2; corresponds to amino acids 14 to 29 of SEQ IDNO:1). The stapled peptide can also include the sequence Phe Ser Asn LeuTrp Arg Leu Leu Pro Gln Asn (SEQ ID NO:5) or the sequence. Gln Ser GlnGln Thr Phe Ser Asn Leu Trp Arg Leu Leu Pro Gln Asn (SEQ ID NO:6). Inthese sequence as in SEQ ID NO:1, 2, 3 and 4), the side chains of twoamino acids separated by 2, 3, 4 or 6 amino acids can be replaced by thelinking group R₃. For example, in SEQ ID NO:5, the side chains of Serand Pro can be replaced by the linking group R₃.

The stapled polypeptide can include all or part (e.g., at least 10, atleast 11, at least 12, at least 13) of the following amino acidsequence:Xaa₁Ser₂Gln₃Xaa₄Thr₅Phe₆Xaa₇Xaa₈Leu₉Trp₁₀Xaa₁₁Leu₁₂Leu₁₃Xaa₁₄Xaa₁₅Asn₁₆(SEQ ID NO:3) wherein each of Xaa₁, Xaa₄, Xaa₇, Xaa₈, Xaa₁₁, Xaa₁₄,Xaa₁₅ are any amino acid (e.g., any of the 20 naturally occurring aminoacids).

In some situations:

Xaa₁=Leu or Gln or the linking group R₃

Xaa₄=Glu or Gln or the linking group R₃

Xaa₇=Ser or the linking group R₃

Xaa₈=Asp or any amino acid other than Asp and Glu (preferably Asn; e.g.,Xaa₈ can be Asp or Asn) or the linking group R₃

Xaa₁₁=Lys or a positively charged amino acid (preferably Arg) or thelinking group R₃

Xaa₁₄=Pro or the linking group R₃

Xaa₁₅=Glu or any amino acid other than Asp and Glu (preferably Gln) orthe linking group R₃.

In some situations, the peptide comprises SEQ ID NO:3 wherein Xaa₁=Leuor Gln or the linking group R₃; Xaa₄=Glu or Gln or the linking group R₃;Xaa₇=Ser or the linking group R₃; Xaa₈=Asp, Asn or the linking group R₃;Xaa₁₁=Lys, Arg or the linking group R₃; Xaa₁₄=Pro or the linking groupR₃; Xaa₁₅=Glu, Gln or the linking group R₃. In the stapled peptides, anyposition occupied by Gln can be Glu instead and any position occupied byGlu can be Gln instead. Similarly, any position occupied by Asn can beAsp instead and any position occupied by Asp can be Asn instead. Thechoice of Asn or Arg and Gln or Glu will depend on the desired charge ofthe stapled peptide.

In some cases the peptide comprises a portion of SEQ ID NO:3 having thesequence:

(SEQ ID NO: 4) Gln₃Xaa₄Thr₅Phe₆Xaa₇Xaa₈Leu₉Trp₁₀Xaa₁₁Leu₁₂Leu₁₃.

Within SEQ ID NO:3, the pairs of amino acid that can be cross-linkedinclude, but are not limited to: the 5^(th) and 12^(th) amino acids;4^(th) and 11^(th) amino acids; 7^(th) and 11^(th) amino acids; and7^(th) and 14^(th) amino acids.

In some instances, the modified peptide binds to HDM2 (e.g., GenBank®Accession No.: 228952; GI:228952) and/or HDM4 (also referred to as HDMX;GenBank® Accession No.: 88702791; GI:88702791). In some instances it canbe useful to create an inactive stapled peptide by replacing one or more(e.g., all three) of Phe₆, Trp₁₀, Leu₁₃ with another amino acid, e.g.,Ala.

Additional useful peptides include non-cross-linked peptides having thefollowing amino acid sequence:Xaa₁Ser₂Gln₃Xaa₄Thr₅Phe₆Xaa₇Xaa₈Leu₉Trp₁₀Xaa₁₁Leu₁₂Leu₁₃Xaa₁₄Xaa₁₅Asn₁₆.(SEQ ID NO:3) wherein each of Xaa₁, Xaa₄, Xaa₇, Xaa₈, Xaa₁₁, Xaa₁₄,Xaa₁₅ are any amino acid (e.g., any of the 20 naturally occurring aminoacids).

In some cases of such non-cross-linked peptides:

Xaa₁=Leu or Gln or the linking group R₃

Xaa₄=Glu or Gln or the linking group R₃

Xaa₇=Ser or the linking group R₃

Xaa₈=Asp or any amino acid other than Asp and Glu (preferably Asn) orthe linking group R₃

Xaa₁₁=Lys or a positively charged amino acid (preferably Arg) or thelinking group R₃

Xaa₁₄=Pro or the linking group R₃

Xaa₁₅=Glu or any amino acid other than Asp and Glu (preferably Gln) orthe linking group R₃

In some cases the non-cross-linked peptide comprises a portion of SEQ IDNO:3 having the sequence:Gln₃Xaa₄Thr₅Phe₆Xaa₇Xaa₈Leu₉Trp₁₀Xaa₁₁Leu₁₂Leu₁₃ (SEQ ID NO:4).

In some instance the modified peptide further comprises, for example:PEG, a fatty acid, or an antibody (e.g., an antibody that targets themodified peptide to a cell expressing p53, HDM2 or HDM4).

In some instances, each w is independently an integer between 3 and 15.In some instances each y is independently an integer between 1 and 15.In some instances, R₁ and R₂ are each independently H or C₁-C₆ alkyl. Insome instances, R₁ and R₂ are each independently C₁-C₃ alkyl. In someinstances, at least one of R₁ and R₂ are methyl. For example R₁ and R₂are both methyl. In some instances R₃ is alkyl (e.g., C₈ alkyl) and x is3. In some instances, R₃ is C₁₁ alkyl and x is 6. In some instances, R₃is alkenyl (e.g., C₈ alkenyl) and x is 3. In some instances x is 6 andR₃ is C₁₁ alkenyl. In some instances, R₃ is a straight chain alkyl,alkenyl, or alkynyl. In some instances R₃ is—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—. In some instances R₃ is—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—. In some instances R₃ is—CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—CH₂—CH₂—CH₂—.

In certain instances, the two alpha, alpha disubstituted stereocenters(alpha carbons) are both in the R configuration or S configuration(e.g., i, i+4 cross-link), or one stereocenter is R and the other is S(e.g., i, i+7 cross-link). Thus, where Formula I is depicted as

the C′ and C″ disubstituted stereocenters can both be in the Rconfiguration or they can both be in the S configuration, for examplewhen x is 3. When x is 6, the C′ disubstituted stereocenter is in the Rconfiguration and the C″ disubstituted stereocenter is in the Sconfiguration. The R₃ double bond may be in the E or Z stereochemicalconfiguration. Similar configurations are possible for the carbons inFormula II corresponding to C′ and C″ in the formula depictedimmediately above.

In some instances R₃ is [R₄—K—R₄]_(n); and R₄ and R₄′ are independentlyalkylene, alkenylene or alkynylene (e.g., each are independently a C1,C2, C3, C4, C5, C6, C7, C8, C9 or C10 alkylene, alkenylene oralkynylene.

In some instances, the polypeptide includes an amino acid sequencewhich, in addition to the amino acids side chains that are replaced by across-link, have 1, 2, 3, 4 or 5 amino acid changes in any of SEQ IDNOs:1-4.

The tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., C₅,C₈ or C₁₁, alkyl or a C₅, C₈ or C₁₁ alkenyl, or C₅, C₈ or C₁₁ alkynyl).The tethered amino acid can be alpha disubstituted (e.g., C₁-C₃ ormethyl). [Xaa]_(y) and [Xaa]_(w) are peptides that can independentlycomprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25 or more contiguous amino acids (preferably 2 or 5contiguous amino acids) of a p53 polypeptide (e.g., any of SEQ IDNOs:1-4) and [Xaa]_(x) is a peptide that can comprise 3 or 6 contiguousamino acids of acids of a p53 peptide.

The peptide can comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50 amino acids of p53 polypeptide. The aminoacids arc contiguous except that one or more pairs of amino acidsseparated by 3 or 6 amino acids are replaced by amino acid substitutesthat form a cross-link, e.g., via R₃. Thus, at least two amino acids canbe replaced by tethered amino acids or tethered amino acid substitutes.Thus, where formula I is depicted as

[Xaa]_(y′), [Xaa]_(x) and [Xaa]_(y″) can each comprise contiguouspolypeptide sequences from the same or different p53 peptides. The sameis true for Formula II.

The peptides can include 10 (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50 or more) contiguous amino acids of ap53 polypeptide wherein the alpha carbons of two amino acids that areseparated by three amino acids (or six amino acids) are linked via R₃,one of the two alpha carbons is substituted by R₁ and the other issubstituted by R₂ and each is linked via peptide bonds to additionalamino acids.

In some instances the polypeptide acts as dominant negative inhibitorp53 degradation. In some instances, the polypeptide also includes afluorescent moiety or radioisotope or a moiety that, can chelate aradioisotope (e.g., mercaptoacetyltriglycine or 1, 4, 7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA)) chelatedto a radioactive isotope of Re, In or Y). In some instances, R₁ and R₂are methyl; R₃ is C₈ alkyl, C₁₁ alkyl, C₈ alkenyl, C₁₁ alkenyl, C₈alkynyl, or C₁₁ alkynyl; and x is 2, 3, or 6. In some instances, thepolypeptide includes a PEG linker, a tat protein, an affinity label, atargeting moiety, a fatty acid-derived acyl group, a biotin moiety, afluorescent probe (e.g. fluorescein or rhodamine).

Also described herein is a method of treating a subject includingadministering to the subject any of the compounds described herein. Insome instances, the method also includes administering an additionaltherapeutic agent, e.g., a chemotherapeutic agent.

The peptides may contain one or more asymmetric centers and thus occuras racemates and racemic mixtures, single enantiomers, individualdiastereomers and diastereomeric mixtures and geometric isomers (e.g. Zor cis and E or trans) of any olefins present. All such isomeric formsof these compounds are expressly included in the present invention. Thecompounds may also be represented in multiple tautomeric forms, in suchinstances, the invention expressly includes all tautomeric forms of thecompounds described herein (e.g., alkylation of a ring system may resultin alkylation at multiple sites, the invention expressly includes allsuch reaction products). All such isomeric forms of such compounds areincluded as are all crystal forms.

Amino acids containing both an amino group and a carboxyl group bondedto a carbon referred to as the alpha carbon. Also bonded to the alphacarbon is a hydrogen and a side-chain. Suitable amino acids include,without limitation, both the D- and L-isomers of the 20 common naturallyoccurring amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G,H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letterabbreviations)) as well as the naturally occurring and unnaturallyoccurring amino acids prepared by organic synthesis or other metabolicroutes. The table below provides the structures of the side chains foreach of the 20 common naturally-occurring amino acids. In this table the“—” at right side of each structure is the bond to the alpha carbon.

Single Three Amino acid Letter Letter Structure of side chain Alanine AAla CH₃— Arginine R Arg HN═C(NH₂)—NH—(CH₂)₃— Asparagine N AsnH₂N—C(O)—CH₂— Aspartic acid D Asp HO(O)C—CH₂— Cysteine C Cys HS—CH₂—Glutamine Q Gln H₂N—C(O)—(CH₂)₂— Glutamic acid E Glu HO(O)C—(CH₂)₂—Glycine G Gly H— Histidine H His

Isoleucine I Ile CH₃—CH₂—CH(CH₃)— Leucine L Leu (CH₃)₂—CH—CH₂— Lysine KLys H₂N—(CH₂)₄— Methionine M Met CH₃—S—(CH₂)₂— Phenylalanine F PhePhenyl-CH₂— Proline P Pro

Serine S Ser HO—CH₂— Threonine T Thr CH₃—CH(OH)— Tryptophan W Trp

Tyrosine Y Tyr 4-OH-Phenyl-CH₂— Valine V Val CH₃—CH(CH₂)—

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide (without abolishing orsubstantially altering its activity. An “essential” amino acid residueis a residue that, when altered from the wild-type sequence of thepolypeptide, results in abolishing or substantially abolishing thepolypeptide activity.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine).

The symbol “

” when used as part of a molecular structure refers to a single bond ora trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acids. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an alpha di-substituted aminoacid).

The term polypeptide encompasses two or more naturally occurring orsynthetic amino acids linked by a covalent bond (e.g., a amide bond).Polypeptides as described herein include full length proteins (e.g.,fully processed proteins) as well as shorter amino acids sequences(e.g., fragments of naturally occurring proteins or syntheticpolypeptide fragments).

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine. The term “alkyl” refers to a hydrocarbon chain that may be astraight chain or branched chain, containing the indicated number ofcarbon atoms. For example, C₁ C₁₀ indicates that the group may have from1 to 10 (inclusive) carbon atoms in it. In the absence of any numericaldesignation, “alkyl” is a chain (straight or branched) having 1 to 20(inclusive) carbon atoms in it. The term “alkylene” refers to a divalentalkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds ineither Z or E geometric configurations. The alkenyl moiety contains theindicated number of carbon atoms. For example, C₂-C₁₀ indicates that thegroup may have from 2 to 10 (inclusive) carbon atoms in it. The term“lower alkenyl” refers to a C₂-C₈ alkenyl chain. In the absence of anynumerical designation, “alkenyl” is a chain (straight or branched)having 2 to 20 (inclusive) carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkynyl” refers to aC₂-C₈ alkynyl chain. In the absence of any numerical designation,“alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Preferred cycloalkyl groups include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, benzimidazolyl, pyrimidinyl,thiophenyl or thienyl, quinolinyl, indolyl, thiazolyl, and the like. Theterm “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkylsubstituted with a heteroaryl. The term “heteroarylalkoxy” refers to analkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,aziridinyl, oxiryl, thiiryl, morpholinyl, tetrahydrofuranyl, and thelike.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, azido, and cyano groups.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1F Synthesis, Sequence, and Biochemical Analysis of SAH-p53Peptides.

FIG. 1A Non-natural amino acids were synthesized as described andcross-linked by ruthenium-catalyzed ring-closing olefin metathesis. FIG.1B SAH-p53 compounds (SEQ ID NOS 9-19, respectively, in order ofappearance) were generated by stapling the p53₁₄₋₂₉ sequence at theindicated positions. Charge, α-helicity, HDM2 binding affinity, cellpermeability, and cell viability are indicated for each compound. FIGS.1C and 1E Circular dichroism spectra revealed enhancement ofalpha-helicity for SAH-p53 compounds. FIGS. 1D and 1F Fluorescencepolarization assays using FITC-peptides and HDM2₁₇₋₁₂₅ demonstratedsubnanomolar HDM2-binding affinities for select SAH-p53 peptides. Note:UAH-p53-8 is the “unstapled” form of SAH-p53-8.

FIG. 2 SAH-p53-8 Reactivates the p53 Transcriptional Pathway. HDM2overexpressing SJSA-1 cells were exposed to the indicated peptides andWestern analyses for p53, HDM2 and p21 were performed at 8-30 h oftreatment.

FIGS. 3A-3C Reactivation of Apoptosis in SAH-p53-8-treated SJSA-1 Cells.FIG. 3A SAH-p53-8 demonstrated specific, dose-dependent cytotoxicity andapoptosis induction. Cell viability assay of SJSA-1 cells treated withSAH-p53 peptides. FIG. 3B Caspase-3 activation assay of SJSA-1 cellstreated with SAH-p53 peptides. FIG. 3C Comparison of caspase-3activation in SJSA-1, HCT-116 p53^(+/+), and HCT-116 p53^(−/−) cellstreated with SAH-p53-peptides (25 μM).

FIGS. 4A and 4B depict an electrospray mass spectrum (positive ion mode)of peptide SAH-p53-4.

FIG. 5 To determine whether SAH-p53 peptides have increased proteolyticstability, the wild type p53₁₄₋₂₉ peptide and SAH-p53-4 were exposed toserum ex vivo. SAH-p53-4 displayed a serum half-life (t_(1/2)) almostfour times longer than that of the unmodified wild type peptide.

FIG. 6 To determine if SAH-p53 peptides 1-4 were cell permeable, JurkatT-cell leukemia cells were incubated with fluoresceinated p53 peptidesfor 4 hours followed by washing, trypsinization, and FACS analysis toevaluate cellular fluorescence. None of the peptides tested producedcellular fluorescence.

FIG. 7A SJSA-1 cells were treated with FITC-SAH-p53-5 and 4.4 kDaTRITC-dextran for 4 hours. Confocal microscopy revealed co-localizationof FITC-SAH-p53-5 peptide with TRITC-dextran in pinosomes. FIG. 7B Toassess whether the permeability of FITC-SAH-p53-5 wastemperature-dependent, Jurkat T-cell leukemia cells were incubated withfluoresceinated p53 peptides for 4 hours at either 4° C. or 37° C.followed by washing, trypsinization, and FACS analysis to evaluatecellular fluorescence. FIG. 7C To determine the kinetics of cellpermeability, Jurkat T-cell leukemia cells were exposed toFITC-SAH-p53-5 peptide and cellular fluorescence was evaluated by FACSanalysis at successive time points. FITC-SAH-p53-5-treated cellsdisplayed a time-dependent increase in cellular fluorescence. FIGS. 7D-1to 7D-3 SJSA-1 cells were treated with FITC-wild type (FIG. 7D-1),SAH-p53-8 (FIG. 7D-2), and SAH-p53-8_(F19A) (FIG. 7D-3) peptides for 4hours followed by FACS and confocal microscopy analyses. Cellularfluorescence was observed after treatment with FITC-SAH-p53 peptides,but not with FITC-wild type p53 peptide.

FIG. 8 SJSA-1 cells were incubated with FITC-peptides followed by lysisand anti-FITC pull down. Native HDM2 co-immununoprecipitated withFITC-SAH-p53-8 but not with wild-type or mutant SAH-p53-8_(F19A)peptides. Left: silver stained gel; right: Western Blots.

FIG. 9 Annexin V binding as an indicator of apoptosis. RKO cells weretreated with peptides at different doses for 24 hours followed bystaining with propidium iodide and FITC-tagged annexin V. Apoptosisinduction was quantified by FACS and the data analyzed with FloJosoftware.

FIG. 10 Fluorescence polarization binding assay of stapled peptides.Fluoresceinated peptides (5 nM) were incubated with recombinantHDM2₁₇₋₁₂₅ (25 pM-10 μM) at room temperature. Binding activity wasmeasured by fluorescence polarization, and Kd values were obtained bylinear regression.

FIG. 11 Cell viability assay. SJSA-1 osteosarcoma cells were treatedwith different concentrations of SAH-p53-8 alone or in combination withthe chemotherapeutic agent doxorubicin (20 μM) for 24 h. Cell viabilitywas assayed by addition of CellTiter-Glo™ bioluminescence reagent andreading on a plate reader.

FIG. 12 Competition by fluorescence polarization. Fluoresceinated, wildtype p53₁₄₋₂₉ (25 nM) was incubated with recombinant HDM2₁₇₋₁₂₅.Unlabeled SAH-p53s were titrated into the mixture, and displacement ofthe labeled ligand was measured by fluorescence polarization.

FIG. 13 Caspase-3 activation assay. SJSA-1 osteosarcoma cells weretreated with different concentrations of SAH-p53s for 24 h. The cellswere then exposed to a caspase-3 specific substrate (Ac-DEVD-AMC (‘DEVD’disclosed as SEQ ID NO: 7)). Fluorescence as a result of cleavage wasmeasured in a microplate reader. To determine the specificity of theactivity, certain peptides were incubated alongside DEVD-CHO (‘DEVD’disclosed as SEQ ID NO: 7), a substrate known to inhibit caspase-3specifically.

FIGS. 14A-14B Immunohistochemistry on mouse tumor xenografts. Two miceeach containing three SJSA-1-derived tumor xenografts were treated with10 mg kg⁻¹ SAH-p53-8 FIG. 14A or vehicle FIG. 14B every 12 hours for twodays. FIG. 14B Paraffin sections were obtained from the tumor xenograftsand were stained using an α-p53 antibody. p53 deficiency due to HDM2amplification is evidenced in the untreated control FIG. 14B, while p53accumulation near capillaries is seen in the sample treated withSAH-p53-8 FIG. 14A.

FIG. 15 Amino acid sequence of human p53 (GenBank® Accession No.CAA42627; gi:50637 (SEQ ID NO: 1)).

FIGS. 16A and 16B depict sequences of various stapled peptides (SEQ IDNOS 20-53, respectively, in order of appearance).

DETAILED DESCRIPTION

Described herein are internally cross-linked alpha helical domainpolypeptides related to human p53. The polypeptides include a tether(also called a cross-link) between two non-natural amino acids thatsignificantly enhance the alpha helical secondary structure of thepolypeptide. Generally, the tether or cross-link (sometimes referred toas staple) extends across the length of one or two helical turns (i.e.,about 3, 4 or about 7 amino acids). Accordingly, amino acids positionedat i and i+3; i and i+4; or i and i+7 are ideal candidates for chemicalmodification and cross-linking. Thus, for example, where a peptide hasthe sequence . . . Xaa₁, Xaa₂, Xaa₃, Xaa₄, Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₉. . . (wherein “ . . . ” indicates the optional presence of additionalamino acids), cross-links between Xaa₁ and Xaa₄, or between Xaa₁ andXaa₅, or between Xaa₁ and Xaa₈ are useful as are cross-links betweenXaa₂ and Xaa₅, or between Xaa₂ and Xaa₆, or between Xaa₂ and Xaa₉, etc.The polypeptides can include more than one crosslink within thepolypeptide sequence to either further stabilize the sequence orfacilitate the stabilization of longer polypeptide stretches. If thepolypeptides are too long to be readily synthesized in one part,independently synthesized, cross-linked peptides can be conjoined by atechnique called native chemical ligation (Bang, et al., J. Am. ChemSoc. 126:1377).

Described herein are stabilized alpha-helix of p53 (SAH-p53) peptidesthat exhibit high affinity for HDM2, and, in contrast to thecorresponding unmodified p53 peptide, readily enter cells through anactive uptake mechanism. As described below, SAH-p53 treatmentreactivated the p53 tumor suppressor cascade by inducing thetranscription of p53-responsive genes, providing the first example of astapled peptide that kills cancer cells by targeting a transcriptionalpathway.

To design SAH-p53 compounds, we placed synthetic olefinic derivatives atpositions that avoid critical HDM2-binding residues. Hydrocarbon staplesspanning the i, i+7 positions were generated by olefin metathesis (FIG.1A). An initial panel of four SAH-p53 peptides was synthesized, eachcontaining a distinctively localized cross-link (FIG. 1B). To evaluatethe structural impact of installing an all-hydrocarbon i, i+7 staple, weconducted circular dichroism (CD) experiments to determine α-helicity.While the wild type p53 peptide displayed 11% α-helical content in waterat pH 7.0, SAH-p53s 1-4 demonstrated 10-59% α-helicity (FIGS. 1B and1C). Fluorescence polarization binding assays using HDM2₁₇₋₁₂₅ andFITC-labeled derivatives of SAH-p53s 1-4 identified SAH-p53-4 as asubnanomolar interactor, having an affinity for HDM2 almost three ordersof magnitude greater than that of the wild type peptide (FIGS. 1B and 1D). SAH-p53-4 also demonstrated improved proteolytic stability (FIG. 5).

We found that the initial SAH-p53 compounds generated were incapable ofpenetrating intact Jurkat T-cells (FIG. 1B and FIG. 6). We noted thatSAH-p53s 1-4 were negatively charged (−2) at physiological pH. Positivecharge is a characteristic feature of certain classes of cellpenetrating peptides.¹¹ In developing a second generation of compounds,we replaced aspartic and glutamic acids with asparagines and glutaminesto adjust peptide charge and mutated select amino acids previouslyreported to participate in p53 nuclear export (L14Q) and ubiquitylation(K24R)^(4, 12) (FIG. 1B). SAH-p53s 5-8 exhibited a 2-8.5 foldenhancement in α-helical content, retained high binding affinity forHDM2, and demonstrated time- and temperature-dependent cellular uptakeby FACS and confocal microscopy (FIGS. 1B, 1E, 1F and 7). Cell viabilityassays using RKO or SJSA-1 cancer cells exposed to SAH-p53 peptidesindicated that SAH-p53-8, which contained point mutations in bothnuclear export and ubiquitylation sites, was the onlystructurally-stabilized, cell-permeable, and high affinity HDM2 binderthat adversely affected cell viability (FIGS. 1B and 4A).

To determine if HDM2-targeting by SAH-p53-8 could specifically restorenative p53 levels; we treated SJSA-1 cells with wild-type, 8, and8_(F19A) peptides for 8-30 hours and monitored p53 protein levels byWestern analysis (FIG. 2). Cells exposed to SAH-p53-8 demonstratedincreased p53 proteins levels that peaked at 18 hours post-treatment.p53 resuppression by 24 hours correlated with the time-dependentupregulation of HDM2 by p53, consistent with an intact p53-HDM2counter-regulatory mechanism.¹³ SAH-p53-8 likewise induced upregulationof the cyclin-dependent kinase inhibitor p21.¹⁴ p21 upregulation incells treated with 8 was detected at 12 hours, reaching peak levels at18 hours. Baseline levels were restored by 30 hours, consistent withresuppression of native p53. HDM2 and p21 levels were unchanged inSJSA-1 cells treated with wild-type or 8_(F19A), highlighting thespecificity of SAH-p53-8 modulation of the p53 signaling pathway.

To examine whether SAH-p53-8-mediated stabilization of native p53 couldinhibit cancer cells by reactivating the apoptotic pathway, we conductedviability and caspase-3 assays using SJSA-1 cells exposed to wild-type,8, and 8_(F19A) for 24 hours (FIGS. 3A-3C). Whereas the wild-type andS_(F19A) peptides had no effect on cell viability, SAH-p53-8 exhibiteddose-dependent inhibition of SJSA-1 cell viability (EC₅₀=8.8 μM) (FIG.3A). Caspase-3 activation by fluorescence monitoring of the cleavedcaspase-3 substrate Ac-DEVD-AMC¹⁵ (‘DEVD’ disclosed as SEQ ID NO: 7)showed that neither the wild-type nor the 8_(F19A) peptides had anyeffect; however, 8 triggered dose-dependent caspase-3 activation(EC₅₀=5.8 μM) that was blocked by DEVD-CHO (‘DEVD’ disclosed as SEQ IDNO: 7), a specific caspase-3 inhibitor, demonstrating that SAH-p53-8specifically inhibited cell viability by activating apoptosis inHDM2-overexpressing SJSA-1 cells (FIG. 3B). As can be seen from FIG. 3C,the SAH-p53-8 mediated inhibition of cell viability observed in SJSA-1cells was also observed in HCT 116 cells, a colon cancer cell line, butnot in an HCT 116 cell line variant lacking p53 (HCT 116 p53^(−/−)).

The identification of multiple organic compounds and p53 peptidomimeticswith anti-HDM2 activity^(8, 16) holds promise for achieving clinicalbenefit from manipulating the p53 pathway. By generating a stapledpeptide-based HDM2 inhibitor, we have documented an in situ interactionbetween SAH-p53-8 and HDM2 (FIG. 8), confirming that its pro-apoptoticactivity derives from restoration of the p53 pathway.

RKO cells were treated with peptides at different doses for 24 hoursfollowed by staining with propidium iodide and FITC-tagged annexin V.Apoptosis induction was quantified by FACS and the data analyzed withFloJo software. As shown in FIG. 9, p53-SAH-p53-6 caused significantapoptosis.

A fluorescence polarization binding assay was used to assess binding ofpeptides to HDM2₁₇₋₁₂₅. Fluoresceinated peptides (5 nM) were incubatedwith recombinant HDM2₁₇₋₁₂₅ (25 pM-10 μM) at room temperature. Bindingactivity was measured by fluorescence polarization, and KD values wereobtained by linear regression. The results of this analysis are shown inFIG. 10.

The effect of SAH-p53-8 alone or in combination with doxorubicin wasexamined as follows. SJSA-1 osteosarcoma cells were treated withdifferent concentrations of SAH-p53-8 alone or in combination with thechemotherapeutic agent doxorubicin (20 μM) for 24 h. Cell viability wasassayed by addition of CellTiter-Glo™ bioluminescence reagent andreading on a plate reader. The results of this analysis are shown inFIG. 11.

The ability of various SAH-p53s to compete with wild-type p53₁₄₋₂₉ forbinding to HDM2₁₇₋₁₂₅ was assessed as follows. Fluoresceinated, wildtype p53₁₄₋₂₉ (25 nM) was incubated with recombinant HDM2₁₇₋₁₂₅.Unlabeled SAH-p53s were titrated into the mixture, and displacement ofthe labeled ligand was measured by fluorescence polarization. Theresults of this analysis are shown in FIG. 12.

The effect of various peptides on caspase-3 activation was examined asfollows. SJSA-1 osteosarcoma cells were treated with differentconcentrations of SAH-p53s for 24 h. The cells were then exposed to acaspase-3 specific substrate. Fluorescence as a result of cleavage wasmeasured in a microplate reader. To determine the specificity of theactivity, certain peptides were incubated alongside DEVD-CHO (‘DEVD’disclosed as SEQ ID NO: 7), a substrate known to inhibit caspase-3specifically. The results of this analysis are shown in FIG. 13.

α,α-Disubstituted non-natural amino acids containing olefinic sidechains of varying length can synthesized by known methods (Williams etal. 1991 J. Am. Chem. Soc. 113:9276; Sehafmeister et al. 2000 J. Am.Chem Soc. 122:5891). For peptides where an i linked to i+7 staple isused (two turns of the helix stabilized) either one S5 amino acid andone R8 is used or one S8 amino acid and one R5 amino acid is used. R8 issynthesized using the same route, except that the starting chiralauxiliary confers the R-alkyl-stereoisomer. Also, 8-iodooctene is usedin place of 5-iodopentene. Inhibitors are synthesized on a solid supportusing solid-phase peptide synthesis (SPPS) on MBHA resin.

Amino Acid and Peptide Synthesis

In the studies described above, Fmoc-protected α-amino acids (other thanthe olefinic amino acids Fmoc-S₅—OH, Fmoc-R₈—OH, Fmoc-R₈—OH, Fmoc-S₈—OHand Fmoc-R₅—OH),2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU), and Rink Amide MBHA resin were purchasedfrom Novabiochem (San Diego, Calif.). Dimethylformamide (DMF),N-methyl-2-pyrrolidinone (NMP), N,N-diisopropylethylamine (DIEA),trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluoresceinisothiocyanate (FITC), and piperidine were purchased from Sigma-Aldrichand used as supplied. The synthesis of the olefinic amino acids has beendescribed elsewhere.^(1, 2)

The polypeptides in the studies described above were synthesizedmanually using Fmoc solid phase peptide chemistry on Rink amide MBHAresin with loading levels of 0.4-0.6 mmol/g resin. The followingprotocol was used:

1. The Fmoc protective group was removed with 20% piperidine in NMP for30 min.

2. The resin was washed with NMP five times.

3. The subsequent Fmoc-protected amino acid was coupled for 30 min (60min for a cross-linker) using Fmoc-AA (10 equiv., 4 equiv. for across-linker), HCTU (9.9 equiv., 3.9 equiv. for a cross-linker), andDIEA (20 equiv., 7.8 equiv. for a cross-linker).

4. The resin was washed with NMP five times.

5. Repeat from step 1.

All peptides were capped with a β-alanine residue at the N-terminus. CDexperiments make use of peptides that have been acetylated at theN-terminus. The acetylation reaction consisted of deprotection of theFmoc group as outlined above, followed by reaction with acetic anhydrideand DIEA. All other experiments shown make use of fluoresceinatedpeptides at the N-terminus. To this end, the peptides with thedeprotected N-terminus were exposed to fluorescein isothiocyanate in DMFovernight in the presence of DIEA.

The ring-closing metathesis reaction was performed on the N-terminalcapped peptide while still on the solid support in a disposable fittedreaction vessel. The resin was exposed to a 10 mM solution ofbis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride (GrubbsFirst Generation Catalyst) in 1,2-dichloroethane or dichloromethane for2 hours. The catalyst addition and 2 hour metathesis reaction wasrepeated once. The resin-bound peptide was washed with CH₂Cl₂ threetimes and dried under a stream of nitrogen.

The peptide was cleaved from the resin and deprotected by exposure toReagent K (82.5% TFA, 5% thioanisole, 5% phenol, 5% water, 2.5%1,2-ethanedithiol) and precipitated with methyl-tert-butyl ether at 4°C. and lyophilized.

The lyophilized peptides were purified by reverse phase HPLC using a C₁₈column (Agilent). The peptides were characterized by LC-MS and aminoacid analysis. Mass spectra were obtained either by electrospray inpositive ion mode or by MALDI-TOF. A representative LC trace and massspectrum are shown below (FIGS. 4-A and 4-B) and the mass spectral datafor all the compounds are likewise shown below in Table 2.

TABLE 2 Mass spectral data for various polypeptides Calculated CompoundMass Found Mass Method WT p53₁₄₋₂₉ 2033.26 2033.12 [M + H] MALDI-TOFSAH-p53-1 2097.41 2097.14 [M + H] MALDI-TOF SAH-p53-2 2132.40 2132.84[M + Na] MALDI-TOF SAII-p53-3 2089.37 2089.18 [M + Na] MALDI-TOFSAH-p53-4 2140.48 2140.70 [M + H] MALDI-TOF SAH-p53-5 2138.5  2139.0[M + H] ESI SAH-p53-6 2165.5  1083.2 [M/2 + H] ESI SAH-p53-7 2152.4 1077.2 [M/2 + H] ESI SAH-p53-8 2180.5  1112.9 [M/2 + Na] ESISAH-p53-8_(F19A) 2104.4  1052.9 [M + H] ESI unstapled SAH-p53-8 2208.5 2209.1 [M + H] ESI FITC-WT p53₁₄₋₂₉ 2401.59 2402.94 [M + Na] MALDI-TOFFITC-SAH-p53-1 2466.74 2467.29 [M + Na] MALDI-TOF FITC-SAH-p53-2 2479.742479.27 [M + Na] MALDI-TOF FITC-SAH-p53-3 2437.72 2437.31 [M + Na]MALIN-TOF FITC-SAH-p53-4 2509.81 2509.10 [M + Na] MALDI-TOFFITC-SAH-p53-5 2401.59 2402.94 [M + Na] MALDI-TOF FITC-SAH-p53-6 2512.8 1257.2 [M/2 + H] ESI FITC-SAH-p53-7 2499.8  1250.6 [M/2 + H] ESIFITC-SAH-p53-8 2527.8  1286.3 [M/2 + Na] ESI FITC-SAH-p53-8_(F19A)2451.7  1248.5 [M/2 + Na] ESI unstapled FITC- 2555.9  1278.5 [M/2 + Na]ESI SAH-p53-8

Circular Dichroism (CD) Spectroscopy

For circular dichroism (CD) spectroscopy compounds were dissolved in H₂Oto concentrations ranging from 10-50 μM. The spectra were obtained on aJasco J-715 spectropolarimeter at 20° C. The spectra were collectedusing a 0.1 cm pathlength quartz cuvette with the following measurementparameters: wavelength, 185-255 nm; step resolution 0.1 nm; speed, 20 nmmin⁻¹; accumulations, 6; bandwidth, 1 nm. The helical content of eachpeptide was calculated as reported previously.³

Ex Vivo Protease Stability

To assess the protease stability of the peptides, fluoresceinatedpeptides (2.5 μg) were incubated with fresh mouse serum (20 μL) at 37°C. for 0-24 hours. The level of intact fluoresceinated compound wasdetermined by flash freezing the serum specimens in liquid nitrogen,lyophilization, extraction in 1:1 CH₃CN:H₂O containing 0.1% TFA,followed by HPLC-based quantitation using fluorescence detection atexcitation/emission settings of 495/530 nm.

Protein Production and Fluorescence Polarization

Purified HDM2₁₇₋₁₂₅ was prepared as follows. Escherichia coli BL21 (DE3)containing the plasmid encoding HDM2₁₇₋₁₂₅ with an N-terminalhexahistidine tag (SEQ ID NO: 8) and a thrombin cleavage site werecultured in kanamycin- and chloramphenicol-containing Luria Broth andinduced with 0.1 mM isopropyl β-D-thiogalactoside (IPTG). The cells wereharvested after 4 hours by centrifugation for 20 min at 3200 rpm,resuspended in buffer A (20 mM Tris pH 7.4, 0.5 M NaCl) and lysed bysonication. Cellular debris was pelleted by centrifugation for 30minutes at 15,000 rpm, and the supernatant was incubated with Ni-NTAagarose (QIAGEN) for 2 h. The resin was washed with buffer A and elutedwith a gradient of imidazole ranging from 5 mM to 500 mM. The fractionscontaining the eluted protein were concentrated and diluted 1:1 withthrombin cleavage buffer (5 mM CaCl₂, 20 mM Tris pH 7.4, 1 μL ML⁻¹β-mercaptoethanol, and 0.8 U mL⁻¹ thrombin). The cleavage reaction wasincubated overnight at 4° C. The reaction was concentrated to 2 mL andpurified by gel filtration using a G75 column. Purity of the protein wasassessed by SDS-PAGE, FPLC and MALDI-TOF and determined to be >90%. Itsidentity was further confirmed by digestion followed by massspectrometry of the resulting peptide fragments.

Fluoresceinated compounds (L_(T)=5-25 nM) were incubated with HDM2₁₇₋₁₂₅in binding assay buffer (140 mM NaCl, 50 mM, Tris pH 8.0) at roomtemperature. Binding activity was measured by fluorescence polarizationon a Perkin-Elmer LS50B luminescence spectrophotometer using a cuvettecontaining a stirbar or a Spectramax M5 Microplate Reader (MolecularDevices). K_(d) values were determined by nonlinear regression analysisof dose response curves using Prism software 4.0 Graphpad. In the caseof compounds where L_(T)<K_(d) and under the assumption thatL_(T)≈L_(free), binding isotherms were fitted to the equation

$\begin{matrix}{P = {P_{f} + \left\lbrack {\left( {P_{b} - P_{f}} \right) \times \frac{R_{T}}{K_{D} + R_{T}}} \right\rbrack}} & (1)\end{matrix}$

where P is the measured polarization value, P_(f) is the polarization ofthe free fluorescent ligand, P_(b) is the polarization of the boundligand, and R_(T) is the receptor/protein concentration.

With compounds where L_(T)>K_(d), the assumption that L_(T)≈L_(free)does not hold due to ligand depletion. As such, binding isotherms werefitted to the more explicit equation

$\begin{matrix}{P = {P_{f} + {\left( {P_{b} - P_{f}} \right)\left\lbrack \frac{\left( {L_{T} + K_{D} + R_{T}} \right) - \sqrt{\left( {L_{T} + K_{D} + R_{T}} \right)^{2} - {4\; L_{T}R_{T}}}}{2\; L_{T}} \right\rbrack}}} & (2)\end{matrix}$where P is the measured polarization value, P_(f) is the polarization ofthe free fluorescent ligand, P_(b) is the polarization of the boundligand, L_(T) is the total concentration of fluorescent ligand and R_(T)is the receptor/protein concentration.⁴ Each data point represents theaverage of an experimental condition performed in at least triplicate.

Flow Cytometry

Jurkat T-cell leukemia cells were grown in RPMI-1640 (Gibco) medium with10% fetal bovine serum, 100 U mL⁻¹ penicillin, 100 μg mL⁻¹, 2 mMglutamine, 50 mM Hepes pH 7, and 50 μM β-mercaptoethanol. SJSA-1 cellswere cultured in McCoy's 5A media (ATCC) supplemented with 10% fetalbovine serum and 100 U mL⁻¹ penicillin. Jurkat cells (50,000 cells perwell) were treated with fluoresceinated peptides (10 μM) for up to 4hours at 37° C. After washing with media, the cells were exposed totrypsin (0.25%; Gibco) digestion (30 min, 37° C.), washed with PBS, andresuspended in PBS containing 0.5 mg mL⁻¹ propidium iodide (BDBiosciences). Cellular fluorescence and propidium iodide positivity wereanalyzed using a FACSCalibur flow cytometer (Becton Dickinson) andFlowJo software (TreeStar). The identical experiment was performed with30 min pre-incubation of cells at 4° C. followed by 4 hour incubationwith fluoresceinated peptides at 4° C. to assess temperature-dependenceof fluorescent labeling.

Confocal Microscopy

Jurkat T-cell leukemia cells were incubated with fluoresceinatedcompounds for 24 hours at 37° C. After washing with PBS, the cells werecytospun at 600 rpm for 5 minutes onto Superfrost plus glass slides(Fisher Scientific). The cells were fixed in 4% paraformaldehyde, washedwith PBS, incubated with TOPRO-3 iodide (100 nM; Molecular Probes) toconterstain nuclei, treated with Vectashield mounting medium (Vector),and imaged by confocal microscopy (BioRad 1024 or Nikon E800).

In a similar fashion, SJSA-1 osteosarcoma cells (1×10⁵ cells) wereincubated in with fluoresceinated compounds for 24 hours at 37° C. inLab-Tek™-CC2 Chamber Slides (Nunc). After washing with PBS, the cellswere fixed in 4% paraformaldehyde, washed with PBS, and treated withDAPI-containing (nuclear counterstain) Vectashield mounting medium(Vector), coverslipped and imaged by confocal microscopy (BioRad 1024 orNikon E800).

Western Blotting

SJSA-1 osteosarcoma cells (1×10⁶) incubated at 37° C. were treated withp53 peptides (20 μM) in serum-free media for 4 hours, followed by serumreplacement and additional incubation for 4-26 additional hours. Thecells were lysed (20 mM Tris-HCl pH 8.0, 0.8% SDS, 1 mM PMSF, 1 U mL⁻¹benzonase nuclease) and the crude lysates were clarified by briefcentrifugation and total protein concentration was determined by usingthe Pierce BCA protein assay. Aliquots containing 5 μg of total proteinwere run on 4-12% Bis-Tris polyacrylamide gels (Invitrogen). Proteinswere detected by chemiluminescence reagent (Perkin Elmer) usingantibodies specific for p53 (DO-1 clone; Calbiochem), HDM2 (IF2 clone;EMD Biosciences), p21 (EA10 clone; Calbiochem), and β-actin(Sigma-Aldrich).

Cell Viability and Apoptosis High-Throughput Assays

SJSA-1 osteosarcoma cells (4×10⁵ cells per well) were incubated in96-well plates and treated with p53 peptides in serum-free media for 4hours, followed by serum replacement and additional incubation for 20hours. Cell viability was assayed by addition of CellTiter-Glo™bioluminescence reagent (Promega) and reading luminescence in aSpectramax M5 microplate reader (Molecular Devices). The extent ofapoptosis was measured through the detection of caspase-3 activity byexposing the cells to a caspase-3-specific substrate (Oncogene).Fluorescence as a result of substrate cleavage was measured in aSpectramax M5 microplate reader (Molecular Devices).

Co-Immunoprecipitation of FITC-SAH-p53 Peptides and Endogenous HDM2

SJSA-1 osteosarcoma cells (1×10⁶) were treated with FITC-p53 peptides(15 μM) in serum-free media for 4 hours, followed by serum replacementand additional 8 hour incubation. The cells were thoroughly washed withserum-containing media and PBS and exposed to lysis buffer (50 mM TrispH 7.6, 150 mM NaCl, 1% Triton-X100, 1 mM PMSF, 1 U mL⁻¹ benzonasenuclease [EMD Biosciences] and complete protease inhibitor tablet[Roche]) at room temperature. All subsequent steps were all performed at4° C. The extracts were centrifuged, and the supernatants were incubatedwith protein A/G sepharose (50 μL 50% bead slurry per 0.5 mL lysatcs;Santa Cruz Biotechnology). The pre-cleared supernatants (500 μL) werecollected after centrifugation, incubated with 10 μL of goat-anti-FITCantibody (AbCam) for 1.5 h followed by protein A/G sepharose for anadditional 1.5 hours. The immunoprecipitation reactions were pelletedand washed three times with lysis buffer. The precipitated proteins weresuspended in SDS-containing loading buffer, boiled, and the supernatantswere processed by SDS-PAGE on 4-12% Bis-Tris gels (Invitrogen). Theproteins were blotted into Immobilon-P membranes (Millipore). Afterblocking, the blots were incubated with either a 1:100 dilution of mouseanti-human HDM2 antibody (IF2 clone; EMD Biosciences) or a 1:200dilution rabbit anti-FITC antibody (Zymed) in 3% BSA in PBS followed byanti-mouse or anti-rabbit horseradish peroxidase-conjugated IgG(Pharmingen). The HDM2 protein and FITC peptides were visualized usingthe Western Lightning™ chemiluminescence reagent (Perkin Elmer) andexposing to film. The gels were stained using a silver stain kit(Bio-Rad) following manufacturer's instructions.

Polypeptides

In some instances, the hydrocarbon tethers (i.e., cross links) describedherein can be further manipulated. In one instance, a double bond of ahydrocarbon alkenyl tether, (e.g., as synthesized using aruthenium-catalyzed ring closing metathesis (RCM)) can be oxidized(e.g., via epoxidation or dihydroxylation) to provide one of compoundsbelow.

Either the epoxide moiety or one of the free hydroxyl moieties can befurther functionalized. For example, the epoxide can be treated with anucleophile, which provides additional functionality that can be used,for example, to attach a tag (e.g., a radioisotope or fluorescent tag).The tag can be used to help direct the compound to a desired location inthe body or track the location of the compound in the body.Alternatively, an additional therapeutic agent can be chemicallyattached to the functionalized tether (e.g., an anti-cancer agent suchas rapamycin, vinblastine, taxol, etc.). Such derivitization canalternatively be achieved by synthetic manipulation of the amino orcarboxy terminus of the polypeptide or via the amino acid side chain.Other agents can be attached to the functionalized tether, e.g., anagent that facilitates entry of the polypeptide into cells.

While hydrocarbon tethers have been described, other tethers are alsoenvisioned. For example, the tether can include one or more of an ether,thioether, ester, amine, or amide moiety. In some cases, a naturallyoccurring amino acid side chain can be incorporated into the tether. Forexample, a tether can be coupled with a functional group such as thehydroxyl in serine, the thiol in cysteine, the primary amine in lysine,the acid in aspartate or glutamate, or the amide in asparagine orglutamine. Accordingly, it is possible to create a tether usingnaturally occurring amino acids rather than using a tether that is madeby coupling two non-naturally occurring amino acids. It is also possibleto use a single non-naturally occurring amino acid together with anaturally occurring amino acid.

It is further envisioned that the length of the tether can be varied.For instance, a shorter length of tether can be used where it isdesirable to provide a relatively high degree of constraint on thesecondary alpha-helical structure, whereas, in some instances, it isdesirable to provide less constraint on the secondary alpha-helicalstructure, and thus a longer tether may be desired. Additionally, whileexamples of tethers spanning from amino acids i to i+3, i to i+4; and ito i+7 have been described in order to provide a tether that isprimarily on a single face of the alpha helix, the tethers can besynthesized to span any combinations of numbers of amino acids.

In some instances, alpha disubstituted amino acids are used in thepolypeptide to improve the stability of the alpha helical secondarystructure. However, alpha disubstituted amino acids are not required,and instances using mono-alpha substituents (e.g., in the tethered aminoacids) are also envisioned.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the described herein will be evident to those ofordinary skill in the art. Additionally, the various synthetic steps maybe performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and Sons(1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

The peptides of this invention can be made by chemical synthesismethods, which are well known to the ordinarily skilled artisan. See,for example, Fields et al., Chapter 3 in Synthetic Peptides: A User'sGuide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77.Hence, peptides can be synthesized using the automated Merrifieldtechniques of solid phase synthesis with the α-NH₂ protected by eithert-Boc or Fmoc chemistry using side chain protected amino acids on, forexample, an Applied Biosystems Peptide Synthesizer Model 430A or 431.

One manner of making of the peptides described herein is using solidphase peptide synthesis (SPPS). The C-terminal amino acid is attached toa cross-linked polystyrene resin via an acid labile bond with a linkermolecule. This resin is insoluble in the solvents used for synthesis,making it relatively simple and fast to wash away excess reagents andby-products. The N-terminus is protected with the Fmoc group, which isstable in acid, but removable by base. Any side chain functional groupsare protected with base stable, acid labile groups. Longer peptidescould be made by conjoining individual synthetic peptides using nativechemical ligation. Alternatively, the longer synthetic peptides can besynthesized by well known recombinant DNA techniques. Such techniquesare provided in well-known standard manuals with detailed protocols. Toconstruct a gene encoding a peptide of this invention, the amino acidsequence is reverse translated to obtain a nucleic acid sequenceencoding the amino acid sequence, preferably with codons that areoptimum for the organism in which the gene is to be expressed. Next, asynthetic gene is made, typically by synthesizing oligonucleotides whichencode the peptide and any regulatory elements, if necessary. Thesynthetic gene is inserted in a suitable cloning vector and transfectedinto a host cell. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. Thepeptide is purified and characterized by standard methods. The peptidescan be made in a high-throughput, combinatorial fashion, e.g., using ahigh-throughput multiple channel combinatorial synthesizer availablefrom Advanced Chemtech.

In the modified polypeptides one or more conventional peptide bondsreplaced by a different bond that may increase the stability of thepolypeptide in the body. Peptide bonds can be replaced by: aretro-inverso bonds (C(O)—NH); a reduced amide bond (NH—CH₂); athiomethylene bond (S—CH₂ or CH₂—S); an oxomethylene bond (O—CH₂ orCH₂—O); an ethylene bond (CH₂—CH₂); a thioamide bond (C(S)—NH); atrans-olefin bond (CH═CH); a fluoro substituted trans-olefin bond(CF═CH); a ketomethylene bond (C(O)—CHR) or CHR—C(O) wherein R is H orCH₃; and a fluoro-ketomethylene bond (C(O)—CFR or CFR—C(O) wherein R isH or F or CH₃.

The polypeptides can be further modified by: acctylation, amidation,biotinylation, cinnamoylation, famesylation, fluoresceination,formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyror Thr), stearoylation, succinylation and sulfurylation. Thepolypeptides of the invention may also be conjugated to, for example,polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight orbranched alkyl groups); fatty acid radicals; and combinations thereof.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with reduced p53 activity. This isbecause the polypeptides are expected to act as inhibitors of p53binding to HDM2 and/or HDMX. As used herein, the term “treatment” isdefined as the application or administration of a therapeutic agent to apatient, or application or administration of a therapeutic agent to anisolated tissue or cell line from a patient, who has a disease, asymptom of disease or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease, the symptoms of disease or thepredisposition toward disease. A therapeutic agent includes, but is notlimited to, small molecules, peptides, antibodies, ribozymes andantisense oligonucleotides.

The polypeptides described herein can be used to treat, prevent, and/ordiagnose cancers and neoplastic conditions. As used herein, the terms“cancer”, “hyperproliferative” and “neoplastic” refer to cells havingthe capacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. Hyperproliferativeand neoplastic disease states may be categorized as pathologic, i.e.,characterizing or constituting a disease state, or may be categorized asnon-pathologic, i.e., a deviation from normal but not associated with adisease, state. The term is meant to include all types of cancerousgrowths or oncogenic processes, metastatic tissues or malignantlytransformed cells, tissues, or organs, irrespective of histopathologictype or stage of invasiveness. “Pathologic hyperproliferative” cellsoccur in disease states characterized by malignant tumor growth.Examples of non-pathologic hyperproliferative cells includeproliferation of cells associated with wound repair.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, or metastatic disorders. Thecompounds (i.e., polypeptides) can act as novel therapeutic agents forcontrolling osteosarcomas, colon cancer, breast cancer, T cell cancersand B cell cancer. The polypeptides may also be useful for treatingmucoepidermoid carcinoma, retinoblastoma and medulloblastoma. Thecompounds can be used to treat disorders associated with unwantedproliferation of cells having reduced activity and/or expression of p53,particularly where the cells produce at least some active p53.

Examples of proliferative disorders include hematopoictic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Exemplary disorders include: acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional exemplary myeloid disorders include, but are notlimited to, acute promyeloid leukemia (APML), acute myelogenous leukemia(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L.(1991) Crit Rev. in Oncol./Hemotol. 11:267-97); lymphoid malignanciesinclude, but are not limited to acute lymphoblastic leukemia (ALL) whichincludes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia(CLL), prolymphocytic leukemia (PLL), multiple mylenoma, hairy cellleukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additionalforms of malignant lymphomas include, but are not limited to non-Hodgkinlymphoma and variants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Pharmaceutical Compositions and Routes of Administration

As used herein, the compounds of this invention, including the compoundsof formulae described herein, are defined to include pharmaceuticallyacceptable derivatives or prodrugs thereof. A “pharmaceuticallyacceptable derivative or prodrug” means any pharmaceutically acceptablesalt, ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.Particularly favored derivatives and prodrugs are those that increasethe bioavailability of the compounds of this invention when suchcompounds are administered to a mammal (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood) orwhich enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies. Preferred prodrugs include derivatives where a group whichenhances aqueous solubility or active transport through the gut membraneis appended to the structure of formulae described herein.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central-nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate,heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate,nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate,salicylate, succinate, sulfate, tartrate, tosylate,trifluoromethylsulfonate, and undecanoate. Salts derived fromappropriate bases include alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N-(alkyl)₄ ⁺ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersible products may be obtained by such quaternization.

The compounds of the formulae described herein can, for example, beadministered by injection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.001 toabout 100 mg/kg of body weight, or according to the requirements of the,particular drug. The, methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositions ofthis invention will be administered from about 1 to about 6 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. A typical preparation will contain from about 5%to about 95% active compound (w/w). Alternatively, such preparationscontain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. Patients may,however, require intermittent treatment on a long-term basis upon anyrecurrence of disease symptoms.

Pharmaceutical compositions of this invention comprise a compound of theformulae described herein or a pharmaceutically acceptable salt thereof;an additional agent including for example, morphine or codeine; and anypharmaceutically acceptable carrier, adjuvant or vehicle. Alternatecompositions of this invention comprise a compound of the formulaedescribed herein or a pharmaceutically acceptable salt thereof; and apharmaceutically acceptable carrier, adjuvant or vehicle. Thecompositions delineated herein include the compounds of the formulaedelineated herein, as well as additional therapeutic agents if present,in amounts effective for achieving a modulation of disease or diseasesymptoms. The term “pharmaceutically acceptable carrier or adjuvant”refers to a carrier or adjuvant that may be administered to a patient,together with a compound of this invention, and which does not destroythe pharmacological activity thereof and is nontoxic when administeredin doses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disoditun hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, may also beadvantageously used to enhance delivery of compounds of the formulaedescribed herein.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art. When the compositions of this invention comprise a combinationof a compound of the formulae described herein and one or moreadditional therapeutic or prophylactic agents, both the compound and theadditional agent should be present at dosage levels of between about 1to 100%, and more preferably between about 5 to 95% of the dosagenormally administered in a monotherapy regimen. The additional agentsmay be administered separately, as part of a multiple dose regimen, fromthe compounds of this invention. Alternatively, those agents may be partof a single dosage form, mixed together with the compounds of thisinvention in a single composition.

Modification of Polypeptides

The stapled polypeptides can include a drug, a toxin, a derivative ofpolyethylene glycol; a second polypeptide; a carbohydrate, etc. Where apolymer or other agent is linked to the stapled polypeptide is/can bedesirable for the composition to be substantially homogeneous.

The addition of polyethelene glycol (PEG) molecules can improve thepharmacokinetic and pharmacodynamic properties of the polypeptide. Forexample, PEGylation can reduce renal clearance and can result in a morestable plasma concentration. PEG is a water soluble polymer and can berepresented as linked to the polypeptide as formula:XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y where n is 2 to 10,000 and X is H or aterminal modification, e.g., a C₁₋₄ alkyl; and Y is an amide, carbamateor urea linkage to an amine group (including but not limited to, theepsilon amine of lysine or the N-terminus) of the polypeptide. Y mayalso be a maleimide linkage to a thiol group (including but not limitedto, the thiol group of cysteine). Other methods for linking PEG to apolypeptide, directly or indirectly, are known to those of ordinaryskill in the art. The PEG can be linear or branched. Various forms ofPEG including various functionalized derivatives are commerciallyavailable.

PEG having degradable linkages in the backbone can be used. For example,PEG can be prepared with ester linkages that are subject to hydrolysis.Conjugates having degradable PEG linkages are described in WO 99/34833;WO 99/14259, and U.S. Pat. No. 6,348,558.

In certain embodiments, macromolecular polymer (e.g., PEG) is attachedto an agent described herein through an intermediate linker. In certainembodiments, the linker is made up of from 1 to 20 amino acids linked bypeptide bonds, wherein the amino acids are selected from the 20naturally occurring amino acids. Some of these amino acids may beglycosylated, as is well understood by those in the art. In otherembodiments, the 1 to 20 amino acids are selected from glycine, alanine,proline, asparagine, glutamine, and lysine. In other embodiments, alinker is made up of a majority of amino acids that are stericallyunhindered, such as glycine and alanine. Non-peptide linkers are alsopossible. For example, alkyl linkers such as —NH(CH₂)_(n)C(O)—, whereinn=2-20 can be used. These alkyl linkers may further be substituted byany non-sterically hindering group such as lower alkyl (e.g., C₁-C₆)lower acyl, halogen (e.g., Cl, Br), CN, NH₂, phenyl, etc. U.S. Pat. No.5,446,090 describes a bifunctional PEG linker and its use in formingconjugates having a peptide at each of the PEG linker termini.

Screening Assays

The invention provides methods (also referred to herein as “screeningassays”) for identifying polypeptides, small molecules, or bifunctionalderivatives which bind to HDM2 and/or HDMX.

The binding affinity of polypeptides that bind HDM2 and/or HDMX can bemeasured using the methods described herein, for example, by using atitration binding assay. HDM2 and/or HDMX can be exposed to varyingconcentrations of a candidate compound (i.e., polypeptide) (e.g., 1 nM,10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM) and binding can bemeasured using surface plasmon resonance to determine the Kd forbinding. Additionally, the binding interactions of fluorescently-labeledSAH-p53 peptides to HDM2 and/or HDMX can be used in a competitivebinding assay to screen for and identify peptides, small molecules, orbifunctional derivatives thereof that compete with FITC-SAH-p53peptides, and further calculate Ki values for binding competition.Candidate compounds could also be screened for biological activity invivo. Cell permeability screening assays in which fluorescently labeledcandidate compounds are applied to intact cells, which are then assayedfor cellular fluorescence by microscopy or high-throughput cellularfluorescence detection can also be used.

The assays described herein can be performed with individual candidatecompounds or can be performed with a plurality of candidate compounds.Where the assays are performed with a plurality of candidate compounds,the assays can be performed using mixtures of candidate compounds or canbe run in parallel reactions with each reaction having a singlecandidate compound. The test compounds or agents can be obtained usingany of the numerous approaches in combinatorial library methods known inthe art.

Thus, one can expose HDM2 (e.g., purified MDM2) or HDMX (e.g., purifiedMDMX) purified to a test compound in the presence of a stapled p53peptide and determining whether the test compound reduces (inhibits)binding of the stapled p53 peptide to MDM2 or MDMX. A test compound thatinhibits binding is a candidate inhibitor of the interaction between p53and MDM2 or MDMX (or both). Test compounds can be tested for theirability to inhibit binding to MDM2 and MDMX in order to identifycompounds that are relatively selective for inhibit p53 binding. In somecases, nutlin-3 (CAS 548472-68-0) can be used as a control sincenutlin-3 is a selective inhibitor of p53 binding to HDM2.

OTHER APPLICATIONS

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

REFERENCES

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The invention claimed is:
 1. A method of treating a subject having adisorder with reduced p53 activity, comprising administering to thesubject having the disorder a therapeutically effective amount of apeptide of Formula (I),

or pharmaceutically acceptable salt thereof, wherein: each R₁ and R₂ isindependently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heteroarylalkyl, or heterocyclylalkyl; each R₃ is independentlyalkylene, alkenylene, alkynylene, or [R₄—K—R₄′]_(n), each of which isindependently substituted with 0-6 R₅; each R₄ and R₄′ is independentlyalkylene, alkenylene or alkynylene; each R₅ is independently halo,alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, a fluorescent moiety,or a radioisotope; each K is independently O, S, SO, SO₂, CO, CO₂,CONR₆, or

each R₆ is independently H, alkyl, or a therapeutic agent; each n isindependently an integer from 1-4; each x is independently 6; each y isindependently an integer from 0-100; each w is independently an integerfrom 0-100; z is an integer from 1-10; and each Xaa is independently anamino acid, wherein the peptide comprises a span of 8 contiguous aminoacid residues that comprises Phe, Leu, and Trp, wherein the peptideexhibits a binding affinity for HDM2 that is from about 0.75 nM to about110 nM.
 2. The method of claim 1, wherein the peptide binds to HDM2. 3.The method of claim 1, wherein each R₃ is independently an alkenylenegroup.
 4. The method of claim 1, wherein each R₃ is independently analkylene group.
 5. The method of claim 1, wherein each R₁ and each R₂ isindependently alkyl.
 6. The method of claim 1, wherein each R₁ and eachR₂ is independently methyl.
 7. The method of claim 1, wherein each R₁and each R₂ is independently H.
 8. The method of claim 1, wherein zis
 1. 9. The method of claim 1, wherein each w is independently aninteger from 3 to
 15. 10. The method of claim 1, wherein each y isindependently an integer from 3 to
 15. 11. The method of claim 1,wherein the peptide is permeable to a cell membrane.
 12. The method ofclaim 1, wherein the peptide comprises a helix.
 13. The method of claim1, wherein the peptide comprises an α-helix.
 14. The method of claim 1,wherein R₃ extends across one helical turn.
 15. The method of claim 1,wherein R₃ extends across two helical turns.
 16. The method of claim 1,wherein the disorder is a cancer selected from the group consisting ofosteosarcoma, colon cancer, breast cancer, T-cell cancer, B-cell cancer,mucoepidermoid carcinoma, retinoblastoma, lymphoma, leukemia, myeloma,and medulloblastoma.
 17. A method for preparing a peptide of Formula(I),

or a pharmaceutically acceptable salt thereof, wherein: each R₁ and R₂is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl,heteroarylalkyl, or heterocyclylalkyl; each R₃ is independentlyalkylene, alkenylene, alkynylene, or [R₄—K—R₄′]_(n), each of which isindependently substituted with 0-6 R₅; each R₄ and R₄′ is independentlyalkylene, alkenylene or alkynylene; each R₅ is independently halo,alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, a fluorescent moiety,or a radioisotope; each K is independently O, S, SO, SO₂, CO, CO₂,CONR₆, or

each R₆ is independently H, alkyl, or a therapeutic agent; each n isindependently an integer from 1-4; each x is independently 6; each y isindependently an integer from 0-100; each w is independently an integerfrom 0-100; z is an integer from 1-10; and each Xaa is independently anamino acid, wherein the peptide comprises a span of 8 contiguous aminoacid residues that comprises Phe, Leu, and Trp, wherein the peptideexhibits a binding affinity for HDM2 that is from about 0.75 nM to about110 nM; the method comprising: (a) synthesizing a peptide or apharmaceutically acceptable salt thereof using solid phase peptidechemistry, wherein the synthesized peptide or pharmaceuticallyacceptable salt thereof comprises at least two moieties capable ofundergoing a metathesis reaction; and (b) performing a ring-closingmetathesis reaction; wherein the peptide of Formula (I) or thepharmaceutically acceptable salt thereof is produced.